HIGHLY CONCENTRATED FORMULATIONS OF SOLUBLE Fc RECEPTORS

ABSTRACT

The present invention relates to novel formulations of soluble Fc receptors and especially to formulations containing high concentrations of soluble Fc RIIB receptor. The invention further relates to the use of such formulations as pharmaceutical compounds for the treatment of autoimmune diseases, infections and other conditions where the immune system is involved.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/787,097, filed Oct. 26, 2015, which is a 371 of PCT/EP2014/001029,filed Apr. 16, 2014, which claims the benefit of EP Patent ApplicationNo. 13002211.4, filed Apr. 26, 2013. The entire contents of each ofthese applications are incorporated by reference herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the determination of denaturing pH range. 0.5 mg/mL husFc

RIIB in 20 mM histidine, 150 mM NaCl (o) and blank buffer (+) wereincubated at the respective pH for 3 h at room temperature. An increasein Sypro Orange fluorescence indicated the presence of denatured husFc

RIIB. husFc

RIIB did not unfold from pH 5.2 to at least pH 11.

FIG. 2 depicts the crystallization of husFc

RIIB. husFc

RIIB crystallization in 10 mM histidine pH 6.7 (FIG. 2A) or 10 mMcitrate pH 5.5 (FIG. 2B) as a function of NaCl and sugar (2:1sucrose:mannitol) concentration. husFc

RIIB crystallizes more readily and the crystal yield is much higher inthe presence of 10 mM histidine, 10 mM NaCl pH 6.7 compared to 10 mMcitrate, 10 mM NaCl pH 5.5.

FIG. 3 depicts husFc

RIIB crystallization performed in 10 mM histidine, 10 mM NaCl as afunction of pH. husFc

RIIB in 10 mM histidine, 10 mM NaCl pH 5.5 was concentrated to 140 mg/mLby ultrafiltration and diluted to 40 mg/mL with appropriate stocksolutions. The crystal yield was determined by measuring the husFc

RIIB concentration in the supernatant after 3 d at 2.8′° C. Eachsolution contained 0.01% polysorbate 20.

FIG. 4 depicts melting temperature Tm of citrate buffered husFc

RIIB formulations measured by Differential Scanning Fluorimetry. Use of0.5 mg/mL husFc

RIIB in 10 mM citrate, 4.5% sugar (2:1 (w/w) sucrose:mannitol), 75 mMNaCl at the indicated pH (FIG. 4A and FIG. 4C), or 0.5 mg/mL husFc

RIIB in 10 mM citrate pH 7.0 supplemented with the indicated amount ofsugar (2:1 (w/w) sucrose:mannitol) and salt (FIG. 4B and FIG. 4D). husFc

RIIB formulations were heated in a 96 well microtiter plate at 1° C./minin the presence of Sypro Orange and the fluorescence emission at 610 nmwas recorded. The fluorescence vs. temperature plots (FIG. 4A and FIG.4B) and their first derivatives (FIG. 4C and FIG. 4D) are shown. Thefirst maximum in the dF/dT plots was defined as husFc

RIIB melting temperature.

FIG. 5 depicts the change of husFc

RIIB melting temperature as a function of pH, sugar and saltconcentration in 10 mM citrate. The average and standard deviation fromthree independent wells are shown.

FIG. 6 depicts turbidity of citrate buffered husFc

RIIB formulations. Optical density at 360 nm was measured as a functionof husFc

RIIB concentration and sucrose concentration after 1 h (FIG. 6A), 12 h(FIG. 6B) and 7 d (FIG. 6C). All formulations contained 10 mM citrate pH7.0, 25 mM NaCl. The optical density of a buffer control was subtracted.At 40% sucrose, the highest concentrated formulation contained only 60mg/mL husFc

RIIB and not 80 mg/mL.

FIG. 7 depicts accelerated stability of citrate buffered husFc

RIIB formulations at 40° C. The increase in optical density at 360 nmwas measured at 10 mg/mL husFc

RIIB in 10 mM citrate, 150 mM NaCl pH 7.0 supplemented with 10%/292 mMsucrose (FIG. 7A, Δ), 10%/292 mM trehalose (FIG. 7A,

), 5%/274 mM mannitol (FIG. 7A, ◯), 30%/876 mM sucrose (FIG. 7B, Δ),30%/876 mM trehalose (FIG. 7B,

), 15%/822 mM mannitol (FIG. 7B, ◯). The buffer control supplementedwith 20% sucrose is also shown (•).

FIG. 8 depicts determination of free polysorbate 20 in the presence ofhusFc

RIIB. husFc

RIIB in 10 mM citrate, 25 mM NaCl, 3% sucrose, 1.5% mannitol, 0.005%polysorbate 20, pH 6.7 was incubated for 1 h at 25° C. (native husFc

RIIB) and 60° C. (denatured husFc

RIIB). After husFc

RIIB was removed by cation exchange chromatography (CEX), the amount ofpolysorbate 20 was measured (FIG. 8A). The blank represents bufferwithout added husFc

RIIB or detergent. The system suitability tests (SST) represent bufferwith 0.005% polysorbate 20, SST2 was CEX treated and SST1 not. Themeasured polysorbate content at 0.08-0.72 mg/mL husFc

RIIB was extrapolated to husFc

RIIB concentrations above 10 mg/mL by linear regression (R2>0.998) ofthe polysorbate concentration versus the logarithmic husFc

RIIB concentration (FIG. 8B).

FIG. 9 depicts solution viscosity of husFc

RIIB in 10 mM citrate, 25 mM NaCl, pH 7.0 was measured at 20° C. (◯) wasfitted to an exponential growth function (−, R2>0.992) and found thatthe solution viscosity of the formulation is low enough to run economicTFF processes up to at least 210 mg/mL.

FIG. 10 depicts particulate load of selected formulations as determinedby fluorescence microscopy. All formulations contained 5 mM citrate pH6.7 and the indicated amount of salt, sugar and detergent. Theformulations were lyophilized and reconstituted to the indicated husFc

RIIB content.

DESCRIPTION

The present invention relates to novel formulations of soluble Fcreceptors and especially to formulations containing high concentrationsof a soluble Fc

RIIB receptor. The invention further relates to the use of suchformulations as pharmaceutical compositions for the treatment ofautoimmune diseases, infections, tumors and other conditions where theimmune system is involved.

Human soluble Fc

RIIB is a promising candidate substance for treatment of IdiopathicThrombocytopenic Purpura, Systemic Lupus Erythematosus and otherautoimmune diseases. It is one of a plurality of soluble antibodyreceptors which have been developed over the past 10 to 15 years.

WO 00/32767 describes soluble Fc receptors (sFcRs) which are composed ofonly the extracellular part of the receptor and are not glycosylated.Due to the absence of the transmembrane domain and the signal peptide,these proteins are present in a soluble form and not bound to cells asis normally the case for Fc receptors (FcRs). Furthermore the sFcRsdescribed in WO 00/32767 can be produced recombinantly and have beensuggested for the treatment of autoimmune diseases due to their abilityto bind the Fc part of antibodies without interfering with othercomponents of the immune system. WO 00/32767 additionally describes thecrystal structure of certain sFcRs and the possibility of developingsubstances that inhibit the interaction of IgG with sFcRs with the aidof these crystal structures. The elucidation of the crystal structureenables finding such inhibitors by e.g. screening the databases usingavailable computer programs or by computer-aided drug design.

The invention which was defined in WO 03/043648 further developed thefindings of WO 00/32767 and provides treatment methods especially fordiseases like multiple sclerosis (MS), systemic lupus erythematosus(SLE), and rheumatoid arthritis (RA) and also for diseases with anelevated level of natural killer (NK) cells. Even if said receptors wereproduced recombinantly in prokaryotes and therefore were unglycosylated,the inventors of WO 03/043648 surprisingly found that although theunglycosylated proteins were expected to be poorly soluble, thereceptors could be purified with relatively high concentrations of up to50 mg/ml of sFc

R in a soluble form.

WO 00/32767, WO 03/043648 and other publications imply an important rolefor FcRs in defense reactions of the immune system. When pathogens haveentered the blood circulation they are bound by immunoglobulins, alsoknown as antibodies. Since the immune response to a pathogen ispolyclonal, a multitude of antibodies are produced and bind to apathogen, leading to the formation of an immune-complex (IC). ICs aresubsequently phagocytised by specialized effector cells (e.g. phagocytesor macrophages) of the immune system and thus removed from thecirculation. The phagocytosis is mediated by the binding of the Fc-partof the antibodies, which, together with the pathogen, form the ICs, toFcRs on the aforementioned effector cells. Other effector cells of theimmune system, such as natural killer cells, eosinophils and mast cellsalso carry FcRs on their surface which upon binding of ICs releasestored mediators such as growth factors or toxins that support theimmune response.

The FcRs of these effector cells also function as signal-transducingmolecules that specifically bind immunoglobulins of various isotypesduring the humoral immune response. In addition, FcRs expressed onnatural killer cells play a fundamental role in the destruction ofantibody-coated target cells (“antibody-dependent cell-mediatedcytotoxicity”, ADCC).

However, in addition to the positive effects of FcRs in the defenseagainst pathogens, overshooting reactions caused by the presence ofauto-antibodies in patients may also occur which result in an undesiredstimulation of the immune system which manifests itself especially asinflammatory or autoimmune diseases. Such immune reactions directedagainst the body's own substances remain a major medical problem andalthough there are approaches for treating them, these approaches arenot equally effective in every patient.

All members of the Fc

R-family, i.e. FcRs which are specific for antibodies of the IgG type,are integral membrane glycoproteins, possessing extracellular domainsrelated to a C2-set of immunoglobulin-related domains, having a singlemembrane spanning domain and an intracytoplasmic domain of variablelength. There are three known Fc

receptor forms, designated Fc

RI (CD64), Fc

RII (CD32), and Fc

RIII (CD16). This invention in preferred embodiments specificallyfocuses on Fc

RII (CD32).

Fc

RII proteins are 40 KDa integral membrane glycoproteins which only bindthe complexed IgG in the ICs. These receptors are the most widelyexpressed Fc

Rs, present on all hematopoietic cells, including monocytes,macrophages, B cells, NK cells, neutrophils, mast cells, and platelets.There are three human Fc

RII genes (Fc

RII-a, Fc

RII-b, Fc

RII-c), all of which bind IgG in aggregates or immune complexes.

Inflammation is a process by which the body's white blood cells react toinfection by foreign substances, such as bacteria and viruses. It isusually characterized by pain, swelling, warmth and redness of theaffected tissue. Effector substances known as cytokines andprostaglandins control this process, and are released in an ordered andself-limiting cascade into the blood or affected tissues. The release ofsuch effector substances increases the blood flow to the area of injuryor infection. Some of the effector substances cause a leak of fluid intothe tissues, resulting in swelling. This protective process maystimulate nerves and cause pain. These changes, when occurring for alimited period in the relevant area, work to the benefit of the body.

In autoimmune diseases the patient's immune system has lost the abilityto discriminate between body-own (“self”) and foreign proteins. Inconsequence, antibodies are generated that recognize “self”-proteins andform immune complexes which continuously activate the immune systembecause the “self”-protein is permanently produced and recognized asforeign. This chronic condition can persist for years leading in the endto severe organ damage and possibly to the death of the patient. Thereare many different autoimmune disorders which affect the body in variousways. For example, the brain is affected in individuals with multiplesclerosis, the gut is affected in individuals having Crohn's disease,and the synovium, bone and cartilage of various joints are affected inindividuals suffering from rheumatoid arthritis. As autoimmune disordersprogress, destruction of one or more types of body tissues, abnormalgrowth of an organ, or changes in organ function may result. Theautoimmune disorder may affect a single organ or tissue type or mayaffect multiple organs and tissues. Organs and tissues commonly affectedby autoimmune disorders include red blood cells, blood vessels,connective tissues, endocrine glands (e.g. the thyroid or pancreas),muscles, joints, and the skin.

Examples of inflammatory and/or autoimmune disorders include, but arenot limited to, primary immune thrombocytopenia (ITP), systemic lupuserythematosus (SLE), rheumatoid arthritis (RA), autoimmune haemolyticanaemia (AIHA), diabetes, Pemphigus vulgaris, Hashimoto's thyroiditis,autoimmune inner ear disease myasthenia gravis, pernicious anemia,Addison's disease, dermatomyositis, Sjogren's syndrome, dermatomyositis,multiple sclerosis, Reiter's syndrome, Graves disease, autoimmunehepatitis, familial adenomatous polyposis and ulcerative colitis.

The Fc

Rs can be divided into two general classes according to their functionwhich may be an activating or inhibitory one. The activating receptorsare associated with a cytoplasmic 16 amino acid immunoreceptortyrosine-based activation motif (ITAM) having the consensus sequenceY-X₂-L/I-X₆₋₁₂-Y-X₂-1/L (Barrow and Trowsdale, EuJI, 2006, 36:1646-1653). This motif can be found, for example, in Fc

RIIA. The other class of FcRs are inhibitory receptors which contain a6-amino acid inhibitory motif (ITIM) in the cytoplasmic part of thereceptor having the consensus sequence S/IN/L-X-Y-X₂-IN/L (Barrow andTrowsdale, EuJI, 2006, 36: 1646-1653). An example of such an inhibitoryFcR is Fc

RIIB.

Fc

RIIB (Fc

RIIB) has two inhibitory activities. One of them is dependent on theITIM-motif and occurs when Fc

RIIB is ligated to an ITAM-carrying receptor (e.g. Fc

RIIA) resulting in the inhibition of ITAM-triggered calcium mobilizationand cellular proliferation. The second inhibitory action of Fc

RIIB involves homo-aggregation of the receptor (Fc

RIIB clustering) which delivers a pro-apoptotic signal into thecytoplasm. The pro-apoptotic signal has only been reported in B-cellsand can be blocked by ligation of Fc

RIIB to the B-cell receptor (BCR) (J V Ravetch, S. Bolland, Annu Rev.Immunol. 2001; 19:275-90.

As mentioned above, in WO 03/043648, sFc

RIIB has already been described for use in pharmaceutical preparationswhere relatively high amounts of Fc

receptors can be included in a reasonable volume of a treatment solutionfor e.g. injection into a patient. Soluble Fc

receptors and especially sFc

RIIB has been suggested for the treatment of autoimmune diseases sincethey can bind to antibodies but do not affect other components of theimmune system. The soluble Fc receptors therefore are able to neutralizeantibodies in the blood stream which has an attenuating effectespecially on autoimmune processes. Possible indications that arealready mentioned in WO 03/043648 include soluble Fc

receptors for treatment of multiple sclerosis (MS), systemic lupuserythematosus (SLE) and rheumatoid arthritis (RA) and also for diseaseswith an elevated level of NK cells to avoid the disadvantages of thepreviously used treatment methods for such diseases.

The teaching of WO 03/043648 focuses on the therein discovered fact thatsoluble Fc receptors can be used to form aqueous solutions with aconcentration of up to 50 mg/ml of soluble receptor. For certainapplications such concentrations of active agent do suffice. In recentstudies, however, a therapeutic dose of sFcRs like sFc

RIIB of significantly more than 1 mg/kg body weight of the patient hasbeen established to be beneficial or even necessary for a successfultreatment of autoimmune diseases.

Subcutaneous administration of pharmaceuticals is considered aneffective and relatively uncomplicated and non-onerous method ofdelivering an active agent to a patient. Compared to intravenousinfusion, which requires a more extensive medical equipment and in mostcases administration at a doctor's office or a clinic, subcutaneousadministration can easily be applied even by the patient himself.Subcutaneous administration will also result in a retarded onset ofaction, i.e. an increased half-life and time to maximal concentration.Also, it was found that the maximum plasma concentration is reduced incase of subcutaneous delivery. These effects are based on theadministration beneath the skin of a patient from where the active agentis transported to the bloodstream. Proteins larger than about 16-20 kDaare generally regarded to be taken up primarily by the lymphatic system,which might be an advantage for Fc

Rs as the target B cell population matures and resides in thelymphatics, too (Porter, C. J. H. and Charman, S. A. (2000), J. PharmSci. 89, 297-310).

The subcutaneous administration route, however, is preferably limited byan injection volume of 1.0 ml up to perhaps 1.5 ml per application(Gatlin, L. A. and Gatlin, C. A. B, (1999), Gapta, P. K. & Brazeau, G.A., eds., Interpharm Press, Denver, pp. 401-425). Thus, aqueous sFc

R solutions as known from WO03/043648 could not have been considered forsubcutaneous administration. Rather, for sufficiently highconcentrations of receptor, precipitation and formation of undesirablylarge crystals of the receptors in the aqueous solution and accordinglyclogging of the needles and/or pain at the injection locus had to beexpected from the teaching of this document. Therefore, in spite of allthe anticipated advantages, subcutaneous administration didn't seem apromising route of treatment and rather intravenous injection orinfusion seemed to be the only viable delivery method.

Accordingly, it was one object of the present invention to provide meansand conditions for less complicated and burdensome administration of Fcreceptors to a patient and for aqueous formulations of soluble Fcreceptors, especially of soluble Fc

RIIB, which contain the receptor in a sufficiently high concentration toenable a subcutaneous treatment regimen for autoimmune diseases.

It was a further object of the present invention to provide such aqueousformulations of highly concentrated soluble Fc receptor in aready-to-use form which is stable under usual storage conditions forpharmaceuticals for more than 24 months or, alternatively, in a formwhich allows for long-term storage and easy and straightforwardadjustment and reconstitution prior to use in subcutaneous applications.

These objects were solved according to the present invention byformulations containing a soluble Fc receptor (sFcR) in an aqueousbuffered solution, wherein the concentration of the Fc receptor isgreater than 50 mg/ml and wherein it contains a physiologicallyacceptable buffer substance.

During the research which led to the present invention, it wassurprisingly found that, contrary to the previous expectations, it ispossible to provide soluble Fc receptors dissolved in suitable buffersolutions with a concentration of even much higher than 70 mg/ml andpreferably more than 150 mg/ml, by means of which it is for the firsttime possible to provide sFcRs also in the form of a pharmaceuticalcomposition for the subcutaneous application. The subcutaneousapplication as parenteral mode of application is simpler and fasterapplicable than an intravenous mode of application. As mentioned above,even the patient himself can carry out a subcutaneous application.

A short and usually thin cannula is required for subcutaneousadministration. The highly concentrated formulation of the presentinvention provides the sFcRs in a completely dissolved and homogenousform and at acceptable viscosity that permits the use of these thincannulas or as crystal suspension containing crystals that are smallenough to pass these thin cannulas. Thus, the patients' convenience isconsiderably increased and an administration of the receptors can beeffected by the subcutaneous route.

The inventive formulations as described in the following enable theprovision of soluble FcRs and especially of soluble Fc

RIIB in concentrations of up to a maximum value which is limited mainlyby the increase in viscosity of the solution due to the highconcentration of the Fc receptor. Upon selection of appropriate buffersubstances and adjustment of suitable osmolalities, a stabilization of220 mg/ml sFc

RIIB and more at a physiological pH is enabled within the framework ofthe present invention.

The physiologically acceptable buffer substances, which are usedaccording to the present invention are commonly used buffers for thepharmaceutical application of suitable solutions. Within the frameworkof the present invention, however, it was found that depending on thebuffer substance used, the pH value of the solution has a considerableinfluence on the solubility of the Fc receptors. Depending on the bufferchosen, the pH value has to be adjusted within a determined range andespecially to a particular optimum for achieving the desired solubilityof the Fc receptors, especially if the purpose is to provide aformulation which does not contain any crystalline sFcR.

It was further found that the protein itself is able to sufficiently actas a buffer due to the presence of positively and negatively chargedamino acids. Thus, e.g. by selecting an appropriate pH value based onthe pK-value of the proteins amino acid side chains, it is possible toforgo the addition of a separate buffer substance as long as a suitableamount of protein is present in the solution to act as thephysiologically acceptable buffer substance.

In preferred embodiments of the present invention, as a buffersubstance, the formulation contains one of a histidine buffer, citratebuffer or phosphate buffer. However, it is particularly preferred toprepare the formulation with either a histidine or a citrate buffer. Inboth cases, it was found that the adjustment of the pH value in thesebuffered solutions permits an adaption of the solubility of the Fcreceptors by means of which high concentrations of Fc receptor can bedissolved, however, can also be caused to crystallize by means ofincreasing or decreasing the pH value depending on the buffer substanceused. This possibility of changing between soluble and crystalline formsof sFcR by the mere adaption of the pH value implies considerableadvantages in view of adaptations with regard to the applicability,preservation and storage stability of pharmaceutical compositions.

For example, during marketing authorization procedures, sufficientstorage stabilities of pharmaceuticals have to be proven. In thiscontext, it is essential that stability data of a storage of at least 12months at a temperature of 5° C. be included by the party requesting themarketing authorization. However, it is desirable and advantageous thata storage stability for more than 24 months under correspondingconditions is achieved, whereby in view of the mentioned storageconditions, a substantial amount, preferably 90% of the pharmaceuticalagent still needs to be present in an active form after the expirationof the time.

In this context, the inventive formulations show particular advantages.For instance, they allow to offer highly concentrated sFcR formulationsin liquid form which exclusively contain dissolved Fc receptor and whichshow a high storage stability.

Moreover, the inventive formulations can be subjected to lyophilizationto provide a solid storage form. Such solid forms might even showimproved storage stability as compared to the liquid formulations. Suchformulations are therefore a further subject matter of the presentinvention.

Lyophilisation can be performed in any suitable manner known to theskilled person for lyophilising proteins. Preferably, conditions as mildas possible are used to avoid protein degradation.

From the solid form, ready-to-use liquid formulations can easily berestored by the addition of water for injection, saline or bufferedaqueous solutions to provide the Fc receptor again in completelydissolved form or intermediate steps can be chosen in which thesolubility is adapted as desired.

Also concentrated forms of inventive formulations are a further subjectmatter of the present invention. Such concentrated forms can be obtainedby e.g. removing part of the liquid to below the solubility limit whichresults in formulations containing at least some crystalline receptors.Also from such formulations, the sFcR can be reconstituted to aready-to-use liquid formulation by adding water for injection, saline orbuffered aqueous solutions to the desired concentration of active sFcRand especially to concentrations in which the sFcR is present entirelyin dissolved form.

Due to the pH dependency of the solubility of Fc receptor in theformulations according to the present invention, the formulationsprovide the additional advantage that the Fc receptor can practically becaused to crystallize entirely by means of adapting the pH and can beobtained (e.g. after sedimentation or centrifugation) as concentratedcrystal suspension. The crystals obtainable thereby can be very small(microcrystals), especially depending on the crystallisation conditions.The faster the crystallisation, the smaller the crystals are. Contraryto prior art, the present invention permits the fast and nearlyquantitative transformation of solubilised sFcR into proteinmicrocrystals and vice versa and thus allows to tailor the solubilityproperties of respective solutions and formulations. This is a furtherpossibility to ensure excellent storage stability and in particular theneed for only little storage space for an active agent, which can thenbe converted into an entirely or mainly soluble form by means ofdissolution in a suitable buffered aqueous solution having a suitable pHvalue. This can, for instance, be done immediately prior to itsapplication as a pharmaceutical by means of admixing concentratedcrystal suspension with a suitable buffer. Alternatively amicrocrystalline suspension or formulation might be directlyadministered by subcutaneous application as the present inventionprovides means to transform the receptor into microcrystals, i.e.crystals that are small enough to pass a cannula or thin needle.Compared to highly concentrated liquid formulations, the viscosity ofsuch microcrystal suspensions is much lower and does not riseexponentially with increasing protein concentration.

For the purposes of the present invention and as used above in thecontext of describing the present invention, Fc receptors are consideredas “crystalline” when crystals have an average size of more than 500 μmin diameter, whereas microcrystalline forms contain microcrystals with asize of equal to or less than 500 μm in diameter.

The present invention enables in an unprecedented way to provide solubleFc receptors in high concentrations in different forms suitable forimmediate or future pharmaceutical use. As illustrated above, this canbe effected in a ready-to-use dissolved form or in a solid, e.g.lyophilized form obtained from such solution or in microcrystalline formprecipitated by pH value adaption, which can then be reconstituted byresolubilization so that a formulation results in which the Fc receptoris contained in high concentration in a suitable buffer solution at thedesired pH value.

In preferred embodiments of the present invention, the inventiveformulation contains as an Fc receptor a sFc

receptor. Regarding the possibility of treating autoimmune diseases, thesFc

RII receptors and especially sFc

RIIB have to be considered. A particularly preferred inventiveformulation thus contains the soluble Fc

RIIB receptor in a pharmaceutically applicable solution with a suitablebuffer substance.

For the purposes of the present invention, the Fc

RIIB receptor has a sequence as described in the prior art, especiallyWO 00/32767 and WO 03/043648 or other documents referring especially toFc

RIIB and especially sFc

RIIB. Further, the term is meant to encompass forms of the receptorwhich can differ especially in their N-terminal parts. An especiallypreferred sFc

RIIB protein is shown in SEQ ID NO:1. This sequence contains amethionine residue at the N-terminus, which is e.g. required forprokaryotic expression, however is cleaved off in a major part of theproduced proteins by bacterial mechanisms lateron. Therefore proteinsaccording to SEQ ID NO:1 lacking the N-terminal methionine are alsoencompassed within the present invention as well as mixtures of proteinswith and without the N-terminal Met. Further, depending on theproduction process and the condition of the bacterial production strain,additional changes in the N-terminal five amino acids can occur. E.g. inaddition to methionine also the following residues can be cleaved off ormethionine could be exchanged for another amino acid like norleucine.Therefore also mixtures of all these proteins differing at theN-terminus and originating from production processes using a DNAsequence which encodes for the amino acid sequence of SEQ ID NO:1 areencompassed by the current definition of sFc

RIIB and especially by the term husFc

RIIB.

In further preferred embodiments, Fc

RIIB proteins are considered as encompassed by the present invention aslong as they have an at least 90% identity to the protein of SEQ IDNO:1. For the determination of sequence identity a comparison is made byaligning the sequences in a manner to provide the maximum correspondenceof amino acids. It is especially preferred that differences in theclaimed proteins occur only within the first ten and most preferablywithin the first five amino acids. It is especially preferred that theproteins have an amino acid identity of at least 95% with differencesoccurring within the first five amino acids of SEQ ID NO:1, wherein thedifferences in the amino acids are based on at least one of deletions,substitutions and additions.

In a particularly preferred embodiment, this formulation according tothe present invention contains the sFc receptor and especially the sFc

RIIB receptor in concentrations of greater than 60 mg/ml, morepreferably greater than 60-80 mg/ml, still more preferably greater than80 mg/ml, even more preferably greater than 100 mg/ml and particularlypreferred greater than 150 mg/ml and most preferably even greater than200 mg/ml.

The inventive formulations optionally contain further pharmaceuticallyacceptable substances, which are for example used for the adjustment ofthe ionic strength of the solution and/or promote the solubility andstability of the receptor protein contained therein. Such substances areknown to the skilled person. For the adjustment of the ionic strengththe inventive formulation optionally contains a salt and preferablyNaCl. For the stabilization of the protein, polyols and especiallysugars and sugar alcohols like sucrose or mannitol can be used. Further,the inventive formulation preferably contains detergents which aresuitable for pharmaceutical applications, as for example polysorbates.

Buffer substances are preferably contained in the formulation of thepresent invention in an amount of 0.1 μM to 300 mM. In more preferredembodiments, the physiologically acceptable buffer is present in anamount of 0.1 to 150 mM and especially 1 to 50 mM.

For the adjustment of the osmolality (isotonicity), salts like sodiumchloride are suitable in such amounts which adjust a preferablyphysiological osmolality, at least as far as the formulation is intendedfor the direct administration to a patient. The osmolality of thesolution can be adjusted over a wide range and can be set to between 10mOsm/kg and greater than 600 mOsm/kg without having a considerableeffect on the solubility of the Fc receptor.

Salt, preferably NaCl, is present in the formulation in a concentrationof about 0 to 250 mM, preferably 5 to 200 mM and most preferably 10 to50 mM.

Polyols like sucrose are not necessarily contained in the inventiveformulations, however in preferred embodiments they are present in anamount of at least 1.0% and more preferred at least 2.0%. The preferredupper limit of the amount of polyols is approximately 25%, morepreferably 15% and most preferably 8%. Sugars are known to stabilizeproteins in solution.

Salts and sugars need to be balanced to adapt the osmolality of theformulation, preferably to be isotonic. The more sugar is contained inthe formulation, the less salt can be added and vice versa.

Suitable amounts of detergents, which are preferably used within thecontext of the invention, are 0.001-0.1%, particularly 0.005-0.05%.

As already mentioned above, the findings, which have been obtainedwithin the framework of the present invention, allow the adaption of thesolubility conditions for soluble Fc receptors and especially sFc

RIIB in such a way that for a content of Fc receptor of greater than 50mg/ml, a predetermined provision of the receptors is made possible ineither completely dissolved form or in a microcrystalline form foradministration to a patient. As also mentioned above, it is oftenadvantageous for an administration to a patient to provide as high aconcentration as possible in a completely dissolved form or at leasttransformable into a dissolved form.

For formulations containing microcrystalline sFcRs, an administration tothe patient may also be possible, whereby the microcrystals completelydissolve after administration and the active substance is available withits physiologic effect. For other administrations and also for storage,it can be of advantage to rather keep the receptor in crystalline form,wherein it is especially stable against degradation and thus loss ofactivity.

Accordingly, preferred embodiments of the present invention areformulations, which are completely liquid and wherein the receptor ispresent in solubilized form or in suitable microcrystalline form

An especially preferred formulation of the present invention containsthe soluble Fc

RIIB receptor in a citrate buffered solution and possesses a pH value ofequal to or greater than 6. The pH value is preferably adjusted within arange of 6.0-7.5. In such citrate buffered solutions with aphysiological pH value, the Fc

RIIB receptor is soluble in concentrations of greater than 140 mg/ml.Due to the physiological pH value, such formulation also has theadvantage that it can directly be administered to a patient withoutcausing side effects like pain at the site of administration.

In another preferred embodiment, the soluble Fc

RIIB receptor is contained in a histidine buffered solution with a pHvalue of 5.2-5.9. When using a histidine buffer, sFc

RIIB is soluble in concentrations of more than 100 mg/ml.

At a pH of approximately 6.0, the solubility of the receptor is stillrelatively high, however, crystalline precipitates are beginning to formwhereas at a higher pH, only a substantially lower solubility of thereceptor is observed.

Both formulations described above enable a high concentration ofsolubilized sFc

RIIB receptor, whereby this could be shown for both the mentionedcitrate buffered formulation and for the histidine buffered formulationup to the viscosity limited regimen of approximately 220 mg/ml (seeenclosed examples).

The inventive formulations further have excellent freeze/thaw stabilityproperties and also excellent stabilities at reduced temperatures of 2°C.-8° C. Even the stability at room temperature is excellent for thesesolutions.

The usability of both solutions is both given for a directadministration to the patient and in the production of a lyophilized orhighly concentrated formulation that can contain crystals and isconvenient for storing or generation of injection solutions which aredirectly reconstitutable by the patient.

For the direct administration, as already mentioned above, the citratebuffered solution with a physiological pH value is particularlypreferred.

A particularly preferred further subject of the invention is aformulation which contains the receptor in crystalline form. Suchformulations are preferably embodied as a citrate buffered suspensionwith a pH value of 5.2-5.9 or alternatively as a histidine bufferedsuspension at a pH value of 6.0-7.5. Such suspensions can for examplepreferably be used as storage-stable forms which, for the administrationto the patient, can be transformed to a formulation containing highconcentrations of the solubilized receptor by means of pH adjustment.Additionally, the same can also be concentrated or the receptor beobtained from them by separation of the solution in order to obtain ahighly concentrated crystal suspension. The receptor can be recovered incompletely dissolved form by reconstitution in a suitable buffer at asuitable pH value.

The described inventive formulations and the finding that, using certainbuffer substances, depending on the pH value different solubility levelsof Fc receptors can be realized enable on the one hand the provision ofready-to-use injections for the subcutaneous administration to apatient, or on the other hand the provision of particularlystorage-stable variants containing crystalline Fc receptor. Evenlyophilized or otherwise solid forms of the receptor are provided whichcan be transformed by a simple addition of the suitable solutions intoready-to-use forms containing high concentrations of soluble receptor.

A further subject of the present invention is thus a pharmaceuticalcomposition, which comprises a formulation according to the presentinvention as described above and in which further pharmaceuticallyacceptable excipients and/or adjuvants and/or carriers can be present.In particularly preferred embodiments these pharmaceutical compositionsare directly applicable for the subcutaneous injection of an effectiveamount of a soluble Fc receptor and especially for the treatment ofautoimmune diseases.

In one preferred embodiment, the pharmaceutical composition preferablycontains a sufficient amount of completely dissolved sFc receptor in asuitable buffer substance and at a physiological pH value. Suchpharmaceutical composition is a ready-to-use medicament and can bedirectly applied to the patient. The dissolved soluble receptor can e.g.easily be absorbed into the patient's lymphatic circulation and bedirectly effective there or after transport within the patient's body byblood or body fluid circulation.

Alternatively, the pharmaceutical composition can contain the receptorin a highly concentrated and at least partially microcrystalline orcrystalline form. Diluted as necessary with suitable buffer solutions,the pharmaceutical composition is again particularly suitable for thesubcutaneous injection of an effective amount of Fc receptors.

As already explained above in the context of describing the presentinvention, Fc receptors are considered as “crystalline” when crystalshave an average size of more than 500 μm in diameter, whereasmicrocrystalline forms contain crystals with a size of equal to or lessthan 500 μm in diameter. As far as a direct application to the patientis considered, pharmaceutical compositions containing completelydissolved Fc receptor can of course be used but also pharmaceuticalcompositions containing formulations with the Fc receptor being solelyor partially in microcrystalline form have merit in pharmaceuticalapplications. These microcrystals are small enough to not clog theneedles for subcutaneous application. The use ofmicrocrystals-containing solutions can be beneficial for e.g. delayed orsustained release of the active sFc receptor to the patient's systemand, under certain circumstances, such microcrystalline forms can evenbe considered as preferred pharmaceutical compositions.

Pharmaceutical compositions of the present invention containingmicrocrystalline or crystalline forms of Fc receptors can e.g. beobtained by concentration of the sFc beyond its solubility byconventional concentration techniques like ultrafiltration. It ispossible to maintain the pharmaceutical in liquid form containing acertain amount of crystals or microcrystals. Instead of using amechanical concentration method for obtaining crystals or microcrystals,it is also possible and a preferred embodiment of the present inventionto crystallize the receptor by adjusting the pH to a value wherein thereceptor has a considerably lower solubility. The precipitated crystalsor microcrystals can be separated from the solution and used for storageand subsequent reconstitution or direct administration. The solid formsof receptor formulations are especially storage stable and maintaintheir effectiveness for at least more than 24 months.

In such cases, the pharmaceutical composition is conveniently providedin a pharmaceutical kit format, which in addition to the solid or highlyconcentrated sFcR also comprises the suitable liquid for thereconstitution of an injectable solution. A further subject matter ofthe present invention is therefore a pharmaceutical kit containingcrystalline or lyophilized soluble Fc receptor and a suitablepharmaceutically acceptable liquid like buffer solution or simply waterfor the reconstitution of the injection solution in suitable separatestorage units.

It is especially preferred for an inventive pharmaceutical kit, if sFcRreceptor and buffer solution for the reconstitution of the injectionsolution are provided in suitable devices for simple mixing and wellprotected against contamination. Preferably, the kit contains a buffersolution based on a phosphate buffer, a histidine buffer or a citratebuffer. It is further preferred for the buffer solutions that the pH isadapted to provide optimum solubility for the Fc receptor. In especiallypreferred embodiments of the present invention, the buffer solution is acitrate buffered solution with a pH of above 6, especially 6.1 to 7.5,or a histidine buffered solution with a pH of below 6.0, especially 5.2to 5.9. The citrate buffered solution is the most preferred buffercontained in a pharmaceutical kit according to the present invention.

The amount of buffered solution contained within the pharmaceutical kitis adapted to the amount of solid or concentrated sFcR in the kit.Depending on whether a complete dissolution of the sFc receptor ormaintaining some amount of microcrystals is desired, a correspondingbuffer is selected in a suitable amount of liquid and also the pH of thesolution is adapted according to the teaching concerning sFcR solubilityas provided herein.

A further subject matter of the present invention is the use of theformulations, the pharmaceutical compositions and pharmaceutical kits ofthe present invention for the prevention or treatment of autoimmunediseases. More particularly, the present invention is intended for theuse within the framework of the prevention or treatment of multiplesclerosis, systemic lupus erythematosus, rheumatoid arthritis, primaryimmune thrombocytopenia and autoimmune haemolytic anemia (AIHA).Further, the formulations, pharmaceutical agents and pharmaceutical kitscan be used for the treatment of inflammatory disorders. The inventivesubject matter enable the application of Fc receptors for allindications for which these have already been described or for whichthey are considered to be suitable in the future. The inventiveformulations and pharmaceutical compositions and kits allow especiallythe subcutaneous administration which is very efficient and easilyapplicable to (or by) a patient. The possibility of administeringespecially high amounts and concentrations of Fc receptors is aparticular advantage of the present invention.

The following examples shall further explain the invention and itsadvantageous effects and embodiments.

Example: Development of High Concentrated Liquid husFc

RIIb Formulations Suitable for Subcutaneous Delivery:

1. Materials

The following solutions of husFc

RIIB (soluble human Fc

RIIB receptor having the amino acid sequence as shown in SEQ ID NO. 1)were used as parent material for all experiments:

a) husFc

RIIB 5 mg/ml concentrate for solution for infusion

-   -   5 mg/mL husFc        RIIB in 5.3 mM NaH₂PO₄, 1.94 mM KH₂PO₄, 150 mM NaCl, 2% (w/v)        mannitol, 0.005% polysorbate 20 pH 6.5        b) husFc        RIIB 20 mg/mL concentrate for solution for infusion    -   20 mg/mL husFc        RIIB in 20 mM histidine, 150 mM NaCl, 2% (w/v) sucrose, 1% (w/v)        mannitol, 0.005% polysorbate 20 pH 6.5.

The following chemicals of at least the indicated grades were used:

Name Purity Supplier Citric acid monohydrate p.a. Merck Sodium hydroxidePh. Eur. (≥98%) Carl Roth Sodium chloride Ph. Eur. (≥99%) Carl RothEthanol, abs. Ph. Eur. (≥99.8%) Carl Roth Histidine Ph. Eur. (≥98.5%)Carl Roth Hydrochloric acid, 37% p.a. Carl Roth Sucrose Ph. Eur. (≥99%)Carl Roth Mannitol ACS reagent (≥99%) Fluka Polysorbate 20 (Tween 20)cell culture tested Sigma Aldrich Trehalose for biochemistry Merck

2. Methods

a) husFc

RIIB Content by UV/Vis Spectroscopy

The sample was transferred to a UV micro-cuvette (UV cuvette micro,Plastibrand, Brand) and the absorbance was measured with aSpectrophotometer (Cary 100, Varian) using the respective buffer asblank.

The husFc

RIIB concentration was calculated by the following equation:

husFc

RIIBconc. [mg/mL]=(A₂₈₀−A₃₂₀)×DF×0.64

-   -   DF≡dilution factor        b) husFc        RIIB Buffer Exchange by Cation Exchange Chromatography 1000-1800        mg husFc        RIIB (husFc        RIIB 5 mg/mL concentrate for solution for Infusion) was        carefully diluted with 10 mM citrate/NaOH pH 6.5 until the        conductivity reached 5.0±0.1 mS/cm. The diluted protein was        filtered (0.2 μm Durapore membrane PVDF hydrophil, 47 mm,        Millipore) and loaded at 5.5 mL/min onto a 57 mL SP Sepharose HP        cation exchange (CEX) resin (26×107 mm, GE Healthcare; equals        17.5-31.6 mg husFc        RIIB/mL resin) equilibrated in 10 mM citrate/NaOH, 20 mM NaCl pH        6.5. Bound protein was washed at 5.5 mL/min with 200 mL 10 mM        citrate/NaOH, 20 mM NaCl pH 6.5 and eluted with a 300 mL linear        gradient ranging from 20 mM to 600 mM NaCl in 10 mM citrate/NaOH        pH 6.5. Eluate collection was started after OD₂₈₀ exceeded 250        mAU (AU: adsorbance units) and was stopped after it dropped        below 200 mAU (1 cm path length, Äkta Explorer 100, GE        Healthcare). The column was regenerated with 100 mL 1 M NaCl,        washed with 150 mL MilliQ H₂O (ultrapure water, Millipore Corp.)        and stored until further use in 20% ethanol. After husFc        RIIB content measurement by UV/Vis spectroscopy the eluate was        filtered (Millex 33 mm, 0.2 μm Durapore PVDF (polyvinyliden        fluoride) hydrophil, both from Millipore Corp.), aliquoted,        snap-frozen in liquid nitrogen and stored at ≤−70° C. until use.

The NaCl content was calculated by correlation of the mean conductivityof the eluate to the measured conductivity at 10 mM citrate/NaOH, 20 mMNaCl and 10 mM citrate/NaOH, 600 mM NaCl.

Histidine buffered husFc

RIIB was prepared similarly using 10 mM histidine/HCl as bufferingspecies.

c) pH Stability Screen

A concentrated husFc

RIIB solution in 20 mM histidine, 324 mM NaCl pH 6.5 was adjusted to 0.5mg/mL husFc

RIIB, 20 mM histidine, 150 mM NaCl, 2.5× Sypro Orange (5000× in DMSO,Molecular Probes™, Invitrogen) using suitable stock solutions. The pH ofthe solution was adjusted between pH 4 and pH 12 based on anexperimental titration curve with 0.2 M HCl or 0.2 M NaOH. 40 μL of thesolution were incubated in a sealed 96 well half area well plate(μclear, black, medium binding; Greiner BioOne) for 3 h at 25° C. andassayed for Sypro Orange fluorescence (Excitation 485 nm, Emission 590nm, gain 60, lag time 0 ps, integration time 40 ps; TECAN Spectrofluorplus).

d) Preparation of High Concentrated Formulations

The required NaCl content of histidine or citrate buffered husFc

RIIB (initial concentration approx. 200-300 mM NaCl) was adjusted bydilution with the appropriate buffer (10 mM citrate or 10 mM histidinepH 6.5) and subsequent ultrafiltration (Vivaspin 20, 5 kDa MWCO,Sartorius). In order to keep the processed volumes small, the procedurewas repeated for up to 3 cycles in total. After final dilution, the pHwas measured (pH-meter HI8314, pH-electrode HI1217, Hanna instruments),adjusted with 0.2 M NaOH or 0.2 M HCl in the appropriate buffer and theprotein solution was concentrated. The husFc

RIIB content was measured by means of UV/Vis spectroscopy in triplicate,the husFc

RIIB concentration was adjusted with the appropriate buffer and thesolution was filtered (Ultrafree MC, 0.2 μm Durapore PVDF hydrophil,Millipore).

e) husFc

RIIB solubility screens in 384 well format

The solubility of high concentrated husFc

RIIB solutions in relation to buffering species, pH, salt concentrationand sugar/polyol concentration was assessed in 384 well format (μclear,white, non-binding, Greiner BioOne) using 30 μL per well. The finalformulations were prepared by direct addition of filtered stocksolutions to each well. The following stock solutions were used: 100-195mg/mL husFc

RIIB, 1.5 M NaCl and 30% (w/v) sucrose+15% (w/v) mannitol in either 10mM citrate pH 7.0 or 10 mM histidine pH 5.5. Each solution was suppliedwith 0.01% (w/w) polysorbat 20 and depending on the screen with 10-50 mMNaCl.

The pH of each well was adjusted with 0.5 M-0.75 M HCl or NaOH. Therequired amount of acid or base was calculated based on theoreticaltitration curves assuming that all nine histidine residues of husFc

RIIB (pK_(a)=6.00) provide additional buffering capacity. The plate wascentrifuged (500·g, 1 min), sealed with adhesive tape (microtest tape,permacel, neo-lab) and stored at 5±3° C. in the dark.

The visual appearance of each formulation was assessed by lightmicroscopy (Axiovert 25f, Carl Zeiss) and ranked according to anarbitrary scale (0=no crystals; 1=some crystals, hardly visible; 2=somecrystals clearly visible; 3=more than 30 crystals per well clearlyvisible; 4=incomplete layer of many crystals (well not fully covered);5=full layer of many crystals (well completely covered).

Taken into account dissolved salt, sugar and protein the osmolality ofeach formulation was calculated according to the following equation:

$\underset{i}{\xi_{m}} = {\Sigma \; v_{i}m_{i}F_{m,i}}$

wherein v_(i) is the number of particles formed by the dissociation ofone molecule of the i^(th) solute and m_(i) is the molality of thei^(th) solute. For simplicity the molal osmotic coefficient F_(m,i) foreach solute was assumed to be equal to 1.f) Small Scale Crystallization of husFc

RIIB

10 μL-450 μL husFc

RIIB at 50-140 mg/mL in 10 mM histidine, 10 mM NaCl, 0.01% polysorbate20, pH 5.5 were diluted with appropriate diluents to 40 mg/mL husFc

RIIB in 10 mM histidine, 10 mM NaCl, 0.01% polysorbate 20 in a 1.5 mLpolypropylene reaction tube. The pH was adjusted to 6.5-7.2 by theaddition of 4.38-6.23 Vol % (final volume after addition of diluents)0.3 M NaOH. The required amount of acid or base was calculated based ontheoretical titration curves assuming that all nine histidine residuesof husFc

RIIB (pK_(a)=6.00) provide additional buffering capacity.

g) Differential Scanning Fluorometry

120 μL of each formulation containing 0.5 mg/mL husFc

RIIB were prepared in a 1.5 mL test tube similarly to the proceduredescribed above in 2.e). Sypro Orange (5000× in DMSO, Molecular Probes™,Invitrogen) was added to a final concentration of 2.5× using a 200×stock in the appropriate buffer. 30 μL of each formulation weretransferred in triplicate to a well plate (MicroAmp 96 well opticalreaction plate, Applied Biosystems) and the plate was sealed withadhesive tape (MicroAmp optical adhesive film, Applied Biosystems). Theplate was subjected to a temperature ramp from 19° C. to 90° C. with aslope of 1° C./min and the fluorescence emission at 610 nm was recorded(7300 Real Time PCR system, Applied Biosystems). The fluorescence wasdifferentiated with respect to time, a spline was calculated and thefirst detected maximum was reported as the melting temperature of husFc

RIIB (Origin 8.0, OriginLab).

h) Turbidity Screen in 384 Well Format

husFc

RIIB formulations were prepared in a 1.5 mL test tube similarly to theprocedure described above in 2.3). 30 μL of each formulation weretransferred in duplicate to a 384 well plate (μclear, white,non-binding, Greiner), the plate was sealed with adhesive tape(microtest tape, permacel, neo-lab) and placed in an incubator. Ascontrol the respective placebo solutions were prepared. The turbiditywas measured at 360 nm (Spectrafluor, bandpass filter 360/35 nm, 3flashes, Tecan). To avoid corrupted measurements due to the condensationof water, the plate reader was pre-heated to the assay temperature.

i) Dynamic Viscosity by Pressure Drop Measurement

The dynamic viscosity of husFc

RIIB containing formulations was determined by measuring the pressuredrop as liquid flows through a flow channel (m-Vroc, Rheosense). Foreach measurement 100 μL containing formulation was filled with a 200 μLpipette into the cylinder of a 100 μL gastight syringe (Hamilton). Thesyringe was installed into the rheometer and 80 μL were injected at aflow rate of 50 μL/min and 20° C.

j) Quantitative Polysorbat 20 Assay

The polysorbate content was determined by a modified protocol which isbased on the colorimetric assay first described by Brown and Hayes,(1955) Analyst 80, 755-767. 500 μL of the solution to be analysed wereextracted three times with 500 μL ethylacetate in a 1.5 mL polypropylenetube (VWR). To accelerate the phase separation, the tube was centrifuged(20′000·g, 5 min, 25° C.). The organic supernatants were combined in aHPLC vial (ND9, screw threaded, with conical bottom and PTFE screw cap,VWR) and the solvent was evaporated (25° C., 10 mbar, 0.5 h-1 h). Theresidual solids were suspended in 800 μL reagent solution (100 mMCo(NO₃)₂, 2.63 M NH₄SCN in water) and were extracted with 150 μL CHCl₃.100 μL of the CHCl₃ extract were transferred to a quartz UV microcuvette (Helima), the spectrum was measured from 200-800 nm (8453 diodearray spectrophotometer, Agilent) and the absorbance at 620 nm correctedby the absorbance at 530 nm was recorded. As blank an extract of anequivalent solution containing no polysorbate 20 was used. Each samplewas prepared in duplicate. The polysorbate 20 content was determinedbased on a standard curve from 0 to 0.006% (w/w) polysorbate 20 in therespective buffer.

k) Lyophilisation

59 formulations containing 15-120 mg/mL husFc

RIIB in 5 mM citrate, 10-25 mM NaCl, 2-8% (w/v) sucrose, trehalose ormannitol and 0.005-0.01% (w/v) polysorbate 20 were prepared and 400 μLwere filled into 1.5 mL clear HPLC vials (32×11.6 mm, wide opening,VWR). The vials were subjected to a conservative lyophilisation cycleusing the freeze-drier Epsilon 2-12D FD02 (Martin Christ, Osterrode,Germany). The vacuum during the freeze-drying process was controlled bya MKS Capacitance Manometer. The samples were frozen at −45° C., primarydrying was performed for 15 h at 45° C. to 15° C., 0.12 mbar andsecondary drying was performed for 10 h at 15° C. to 20° C., 0.12 mbar.The lyophilisate was reconstituted in 100-400 μL water for injection.The solution was analyzed in respect to the formation of particulates byvisual inspection, turbidity measurement and fluorescence microscopy. Inbrief, for fluorescence microscopic examination 50 μL of the husFc

RIIB containing solution was placed into a 384 well plate (μclear,white, non-binding, Greiner) and mixed with 5 μL 25× Sypro Orange (5000×in DMSO, Molecular Probes™, Invitrogen) in 5 mM citrate, 10 mM NaCl pH6.7. The plate was incubated for 10 min at 25° C., centrifuged (1′000·g,3 min) and the appearance of each formulation was assessed byfluorescence microscopy (Axiovert 25f, excitation filter 470/20 nm,dichroic 493 nm, emission filter 503-530 nm, Carl Zeiss).

3. Results

a) Definition of pH Range and Buffering Species for High ConcentratedhusFc

RIIB Solutions

The husFc

RIIB denaturing pH range was determined using Sypro Orange as anindicator for the presence of unfolded protein. Sypro Orange is anenvironment sensitive dye whose fluorescence emission is stronglyincreased after its binding to hydrophobic structures (Layton &Hellinga, 2010, Biochemistry 49 (51), 10831-10841). FIG. 1 shows theresults of experiments to determine the denaturing pH range. Therefore,0.5 mg/mL husFc

RIIB in 20 mM histidine, 150 mM NaCl (o) and blank buffer (+) wereincubated at the respective pH for 3 h at room temperature. An increasein Sypro Orange fluorescence indicated the presence of denatured husFc

RIIB. As shown in FIG. 1, husFc

RIIB did not unfold from pH 5.2 to at least pH 11.

To prevent pain during subcutaneous administration the administeredsolution should have a pH in the physiologic range. Typical bufferingspecies that buffer in this range and are generally regarded as safeinclude histidine (pka ˜6.0), citrate (pKa₃˜6.4) and phosphate(pKa₂˜7.2). Due to its tendency to promote pH shifts during freeze/thaw(MacKenzie, 1977) phosphate was not included in subsequent solubilityscreens.

In an initial attempt to determine the limiting husFc

RIIB concentration in respect to protein precipitation husFc

RIIB was concentrated by ultrafiltration in the presence of 10 mMhistidine or 10 mM citrate and 10 mM NaCl until a visible precipitatewas formed. As shown in Table 1 histidine buffered husFc

RIIB showed increased solubility in the slightly acidic range from pH5.5 to 6.0 whereas citrate as buffering species provided solubility inthe near neutral pH range around pH 6.5. In summary the husFc

RIIB solubility at various pH is largely dependent on the bufferingspecies used. By microscopy it was shown that the precipitate iscomposed of crystalline needles.

TABLE 1 husFc 

 RIIB solubility limit (in mg/mL) in 10 mM histidine or 10 mM citrate atlow ionic strength from pH 5.5 to 7.5. husFc 

 RIIB was concentrated by ultrafiltration until the solution becamecloudy. Histidine buffered husFc 

 RIIB precipitated at pH 6.0, 6.5 and 7.0 whereas citrate bufferedhusFc 

 RIIB precipitated at pH 5.5 and pH 6.0. The sediment was identified ashusFc 

 RIIB protein crystals. pH 5.5 6.0 6.5 7.0 Histidine >162 ≤174 ≤40 ≤10Citrate ≤40 ≤158 >154 >148b) Crystallization of husFc

RIIB

Histidine buffered husFc

RIIB remains soluble above 100 mg/mL at pH 5.5 and can be crystallizedby neutralization at low ionic strength, on the contrary citratebuffered husFc

RIIB remains soluble at neutral pH above 100 mg/mL and can becrystallized by mild acidification at low ionic strength (Table 1). In afurther test, crystallisation of husFc

RIIB was investigated in dependence of the presence and the amount ofsugar and NaCl in the buffer. husFc

RIIB crystallisation was performed in 10 mM histidine pH 6.7 (FIG. 2a )or 10 mM citrate pH 5.5 (FIG. 2b ) as a function of NaCl and sugar (2:1sucrose:mannitol) concentration. husFc

RIIB in 10 mM histidine, 10 mM NaCl pH 5.5 or 10 mM citrate, 10 mM NaClpH 7.0, respectively, was concentrated to 140 mg/mL by ultrafiltrationand diluted to 40 mg/mL with appropriate stock solutions. In case ofhistidine buffered husFc

RIIB, the crystal yield was determined by measuring the husFc

RIIB concentration in the supernatant after 3 d at 2-8° C. In case ofcitrate buffered husFc

RIIB, no crystal growth was detected until day 10. Therefore, thecrystal yield was determined after 14 d at 2.8° C. Each solutioncontained 0.01% polysorbate 20.

FIG. 2 shows that husFc

RIIB crystallizes more readily and the crystal yield is much higher inthe presence of 10 mM histidine, 10 mM NaCl pH 6.7 compared to 10 mMcitrate, 10 mM NaCl pH 5.5. Further reduction of the sodium chlorideconcentration from 10 mM to 5 mM resulted in a marginal increase incrystal yield when using histidine as buffering species but showed astrong effect when using citrate buffer. Reduction of the saltconcentration below 5 mM and/or reduction of pH might further stimulatethe crystallization process in the presence of citrate. Increased NaClconcentrations or the addition of polyols, e.g. sucrose or mannitol,above 5% inhibited the crystallization process.

FIG. 3 shows an experiment in which husFc

RIIB crystallisation was performed in 10 mM histidine, 10 mM NaCl as afunction of pH. husFc

RIIB in 10 mM hisitidine, 10 mM NaCl pH 5.5 was concentrated to 140mg/mL by ultrafiltration and diluted to 40 mg/mL with appropriate stocksolutions. The crystal yield was determined by measuring the husFc

RIIB concentration in the supernatant after 3 d at 2.8′° C. Eachsolution contained 0.01% polysorbate 20.

Within a pH range of at least 0.5 units, more than 93% of histidinebuffered husFc

RIIB can be crystallized. With a total husFc

RIIB concentration of 40 mg/mL the solubility limit of husFc

RIIB in 10 mM histidine, 10 mM NaCl pH 6.7-7.2 is below 2.8 mg/mL. At pH6.9 the crystallization was complete in less than one hour at 25° C.

c) husFc

RIIB solubility screens

In order to define so called solubility sweet spots, i.e. conditionswhere husFc

RIIB remains soluble above 100 mg/mL and does not crystallize, varioushusFc

RIIB formulations were prepared in a 384 well microtiter plate andincubated for at least 4 weeks at 2-8° C. Parameters included into thescreen were husFc

RIIB concentration (70, 100, 120, and 150 mg/mL), buffering species(histidine or citrate), pH (5.5, 6.0, 6.5, 7.0, 7.5), NaCl concentration(10-225 mM) and sugar content (0-7.5%). The initial screens at 70 mg/mL(Table 2 and Table 5) and 100 mg/mL husFc

RIIB (Table 3 and Table 6) were conducted with two sugar levels (0% or3%) and four different NaCl concentrations (10, 50, 225 mM).

Basically these prescreens reproduced the results that were alreadyobtained during the above mentioned concentration screen (Example 2a)and crystallization screens (Example 2b). Histidine buffered husFc

RIIB crystallizes above pH 5.5, whereas citrate buffered husFc

RIIB crystallizes at pH 5.5 to 6.0. Increasing NaCl or sugarconcentrations reduced the crystallization process in both cases.

In all subsequent screens at 120 mg/mL and 150 mg/mL husFc

RIIB, only formulations that are isotonic with blood, i.e. with acalculated osmolality around 308 mOsmol/kg were included. For thatreason high sugar concentrations were matched with low concentrations ofNaCl and vice versa. Histidine based formulations with 120 mg/mL husFc

RIIB were able to prevent crystallization of husFc

RIIB under neutral pH and high ionic strength but with one exception allhistidine based formulations at 150 mg/mL husFc

RIIB showed strong crystal growth after 4 weeks at 2-8° C. (Table 4). Onthe other hand, citrate based formulations were stable up to 150 mg/mLhusFc

RIIB from pH 6.5 to 7.5 and at all sugar/salt combinations tested (Table7).

Therefore citrate buffered husFc

RIIB represents the best basis for the development of a highconcentrated liquid formulation suitable for subcutaneous application,i.e. with physiologic pH and tonicity.

d) Thermal Stability of High Concentrated husFc

RIIB Formulations

The formation of non-native protein aggregates and particulates couldpose a major obstacle for the development of a high concentrated proteinformulation (Shire et al., 2010, Chapter 15. High-concentration antibodyformulations. In Formulation and Process Development Strategies forManufacturing Biopharmaceuticals. Jameel, F. & Hershenson, S., eds.,John Wiley, Hoboken, N.J.). For that reason formulation candidates (c.f.Example 2c) were ranked in respect to their ability to preserve husFc

RIIB's native structure in the presence of thermal stress and henceprohibit non-native aggregation.

At first the melting temperature T_(m) of citrate buffered husFc

RIIB formulations was measured by Differential Scanning Fluorimetry.FIG. 4 shows the respective results that were obtained using 0.5 mg/mLhusFc

RIIB in 10 mM citrate, 4.5% sugar (2:1 (w/w) sucrose:mannitol), 75 mMNaCl at the indicated pH (FIGS. 4 (a) and (c)), or 0.5 mg/mL husFc

RIIB in 10 mM citrate pH 7.0 supplemented with the indicated amount ofsugar (2:1 (w/w) sucrose:mannitol) and salt (FIGS. 4 (b) and (d)). husFc

RIIB formulations were heated in a 96 well microtiter plate at 1° C./minin the presence of Sypro Orange and the fluorescence emission at 610 nmwas recorded. The fluoresence vs. temperature plots (a) and (b) andtheir first derivatives (c) and (d) are shown. The first maximum in thedF/dT plots was defined as husFc

RIIB melting temperature.

Depending on the composition of the respective formulation candidateT_(m) values from 50.8° C. to 55.5° C. were measured. The largestinfluence on the melting temperature had the pH with a T_(m) increase byapprox. 3.5° C. when the pH was lowered from 7.5 to 6.5. Addition of7.5% sugar increased the T_(m) by approx. 1° C. (FIG. 5, showing thechange of the husFc

RIIB melting temperature as a function of pH, sugar and saltconcentration in 10 mM citrate. The average and standard deviation fromthree independent wells are shown). Although the NaCl concentration waslowered in parallel the increased thermal stability is clearly afunction of the increased sugar concentration and not the decreased saltcontent, as shown in earlier experiments (data not shown) and accordingto the theory of preferential exclusion (Timasheff, 1992, Chapter 9.Stabilization of Protein Structure. In Stability of ProteinPharmaceuticals, Part B: In vivo pathways of degradation and strategiesfor protein stabilization. Ahern, T. J. & Manning, M. C., eds., PlenumPress, New York, pp. 265-285). This is also exemplified by raising thesugar concentration from 7.5% to 40% which resulted in a T_(m) increaseby 6.2° C.

Next it was determined to what extent the stabilization of husFc

RIIB's secondary and tertiary structure, as indicated by a high meltingtemperature, would inhibit the formation of insoluble proteinaggregates. Therefore the turbidity of citrate buffered husFc

RIIB formulations was measured after incubation at 37° C., a temperaturewell below the measured T_(m). To this purpose, the acceleratedstability of citrate buffered husFc

RIIB formulations at 37° C. were determined. The optical density at 360nm was measured as a function of husFc

RIIB concentration and sucrose concentration after 1 h (a), 12 h (b) and7 d (c). All formulations contained 10 mM citrate pH 7.0, 25 mM NaCl.The optical density of a buffer control was subtracted. At 40% sucrose,the highest concentrated formulation contained only 60 mg/mL husFc

RIIB and not 80 mg/mL.

The results are shown in FIG. 6. With increasing sucrose concentration(increasing concentrations from back to front on z-axis in all diagramsof FIG. 6), i.e. increasing melting temperature of the protein, the risein turbidity is retarded. With no added sucrose all formulations with astrength 0 mg/mL husFc

RIIB became already turbid after one hour at 37° C., whereas at 40%sucrose no significant increase in turbidity up to 60 mg/mL husFc

RIIB was observed even after seven days at 37° C. Above a certainprotein concentration the absolute turbidity increases linearly withincreasing husFc

RIIB concentration but below that threshold the formation of insolubleprotein aggregates is extremely slow or even inhibited. With increasingsucrose concentrations this threshold is shifted to higher husFc

RIIB concentrations, but from the physiologic point of view unacceptablehigh sucrose concentrations will be needed to stabilize husFc

RIIB at 37° C. and a concentration exceeding 60 mg/mL.

A further test was conducted regarding the accelerated stability ofcitrate buffered husFc

RIIB formulations at 40° C. The results are shown in FIG. 7. Theincrease in optical density at 360 nm was measured at 10 mg/mL husFc

RIIB in 10 mM citrate, 150 mM NaCl pH 7.0 supplemented with 10%/292 mMsucrose (a, Δ), 10%/292 mM trehalose (a,

), 5%/274 mM mannitol (a, ◯), 30%/876 mM sucrose (b, Δ), 30%/876 mMtrehalose (b,

), 15%/822 mM mannitol (b, ◯). The buffer control supplemented with 20%sucrose is also shown (•).

Based on these observations different sugars and sugar alcohols wereranked in respect to their ability to suppress the formation ofinsoluble protein aggregates. As shown in FIG. 7, the most efficientstabilizer is sucrose.

e) Definition of Required Detergent Concentration

Turbidity assays (data not shown) indicated in accordance with publisheddata (Timasheff, 1992, Chapter 9. Stabilization of Protein Structure. InStability of Protein Pharmaceuticals, Part B: In vivo pathways ofdegradation and strategies for protein stabilization. Ahern, T. J. &Manning, M. C., eds., Plenum Press, New York, pp. 265-285) thatincreasing detergent concentrations destabilize husFc

RIIB. Also it is speculated that the use of high detergentconcentrations may lead to increased immunogenicity (Hermeling et al.,2003, Pharm. Res. 20, 1903-1907). For that reason it would be mandatoryto keep the detergent concentration as low as possible withoutcompromising its stabilizing effect towards surface stress.

Ideally, the polysorbate 20 concentration would be fixed at 0.005%, theconcentration which is used for already established liquid husFc

RIIB formulations containing 5-20 mg/mL husFc

RIIB. But since the strength of the newly developed formulation will beabove 50 mg/mL and polysorbate 20 may bind to the protein, it wasquestioned whether the detergent concentration must be raised above0.005%.

In order to test the hypothesis of unspecific detergent binding by theprotein a citrate buffered formulation candidate containing 0.005%polysorbate 20 was supplied with increasing concentrations of husFc

RIIB and the solutions were incubated for one hour at room temperatureand 60° C., a temperature well above the determined T_(m) of husFc

RIIB. After the protein and hypothetically bound polysorbate 20 wereremoved by cation exchange chromatography the amount of free polysorbatewas measured. In FIG. 8 an experiment is shown that determines freepolysorbate 20 in the presence of husFc

RIIB. husFc

RIIB in 10 mM citrate, 25 mM NaCl, 3% sucrose, 1.5% mannitol, 0.005%polysorbate 20, pH 6.7 was incubated for 1 h at 25° C. (native husFc

RIIB) and 60° C. (denatured husFc

RIIB). After husFc

RIIB was removed by cation exchange chromatography (CEX), the amount ofpolysorbate 20 was measured (a). The blank represents buffer withoutadded husFc

RIIB or detergent. The system suitability tests (SST) represent bufferwith 0.005% polysorbate 20, SST2 was CEX treated and SST1 not. Themeasured polysorbate content at 0.08-0.72 mg/mL husFc

RIIB was extrapolated to husFc

RIIB concentrations above 10 mg/mL by linear regression (R²>0.998) ofthe polysorbate concentration versus the logarithmic husFc

RIIB concentration (b).

As shown in FIG. 8, a linear relationship between the free polysorbatecontent and the logarithmic husFc

RIIB concentration was established. Therefore it is expected that theconcentration of free polysorbate will only change marginally, byapprox. 0.0001-0.0002%, in case that the strength of the formulation isincreased from 20 to 100 mg/mL. The significantly lower polysorbateconcentration in the samples compared to the control at 0.005% islargely attributed to the fact the samples were diluted with CEXequilibration buffer, which was prepared without polysorbate 20, duringcolumn loading (c.f. FIG. 8 SST 1 vs. SST2).

f) Viscosity of High Concentrated husFc

RIIB Formulations

Highly concentrated protein formulations are characterized by a highviscosity (Shire et al., 2010, supra). Therefore the manufacturabilityof a highly concentrated protein formulation might be hampered by itsviscosity as the concentration process by tangential flow filtration maybecome unacceptable slow. In a further experiment, the solutionviscosity of husFc

RIIB in 10 mM citrate, 25 mM NaCl, pH 7.0 was measured at 20° C. Theexperimental data as shown in FIG. 9 (◯) was fitted to an exponentialgrowth function (−, R²>0.992) and found that the solution viscosity ofthe formulation is low enough to run economic TFF processes up to atleast 210 mg/mL.

g) Lyophilisation of husFc

RIIB Containing Formulations

59 formulations with varying husFc

RIIB, sugar, salt and detergent content were subjected to a conservativelyophilisation cycle. The solids were reconstituted with a volume ofwater for injection that was equal to or less than the original volumeprior to the lyophilisation process. In doing so the husFc

RIIB content after reconstitution was adjusted to a nominal content of60-180 mg/mL. The suitability of the various formulations forlyophilisation was evaluated based on the reconstitution time and theparticulate contamination after reconstitution. Several formulationswere identified that could be reconstituted in less than 2 min andneither showed an increased turbidity nor the formation of particulatein the visible and sub-visible range. Based on the above describedlyophilisation screen it was shown that ideal formulations contained lowamounts of husFc

RIIB (e.g. 15-60 mg/mL) prior to lyophilisation and were reconstitutedwith low volumes of water for injection, thereby increasing the finaldetergent concentration. The particulate load of selected formulationsas determined by fluorescence microscopy is shown in FIG. 10. Allformulations contained 5 mM citrate pH 6.7 and the indicated amount ofsalt, sugar and detergent. The formulations were lyophilized andreconstituted to the indicated husFc

RIIB content.

1. A formulation comprising a soluble Fc receptor (sFcR) at aconcentration of greater than 50 mg/ml.
 2. The formulation of claim 1,further comprising a buffer solution at a concentration of 0.1 μM to 300mM, wherein said buffer solution comprises histidine, citrate, orphosphate.
 3. The formulation of claim 1, wherein said Fc receptor is anFc

receptor.
 4. The formulation of claim 3, wherein the receptor is an Fc

RII receptor.
 5. The formulation according to claim 4, wherein thereceptor is Fc

RIIB
 6. The formulation of claim 1, wherein the concentration of the Fcreceptor is greater than 60 mg/ml.
 7. The formulation of claim 6,wherein the concentration of the Fc receptor is greater than 80 mg/ml.8. The formulation of claim 1, wherein the concentration of the Fcreceptor is greater than 200 mg/ml.
 9. The formulation of claim 5,wherein said formulation further comprises at least one of a sugar, asalt, or a detergent.
 10. The formulation of claim 5, wherein thereceptor is present in dissolved form.
 11. The formulation of claim 5,wherein the receptor is present in a microcrystalline form.
 12. Theformulation of claim 2, wherein it is a citrate buffered solution andthe pH of the formulation is adjusted to from 6.0 to 7.5.
 13. Theformulation of claim 2, wherein it is a histidine buffered solution andthe pH is adjusted to from 5.2 to 5.9.
 14. The formulation of claim 2,wherein it is a citrate buffered solution and its pH is adjusted to from5.2 to 5.9.
 15. The formulation of claim 2, wherein it is a histidinebuffered solution and its pH is adjusted to from 6.0 to 7.5.
 16. Amethod for treating an autoimmune disease, comprising subcutaneouslyadministering to a patient a composition comprising a soluble Fcreceptor (sFcR) at a concentration of greater than 50 mg/ml.
 17. Themethod of claim 16, said composition further comprising a buffersolution at a concentration of 0.1 μM to 300 mM, wherein said buffersolution comprises at least one of histidine, citrate, or phosphate. 18.The method of claim 17, wherein said buffer solution comprises ahistidine buffered solution at a pH from 5.2 to 5.9.
 19. The method ofclaim 17, wherein said buffer solution comprises a citrate bufferedsolution at a pH from 5.2 to 5.9.
 20. The method of claim 17, whereinsaid buffer solution comprises a histidine buffered solution at a pHfrom 6.0 to 7.5.
 21. The method of claim 17, wherein it is a citratebuffered solution and the pH of the formulation is adjusted to from 6.0to 7.5.
 22. The method of claim 17, wherein said subcutaneousadministration comprises administration of a volume of 1.5 mL or lessper application.